Modified green fluorescent proteins

ABSTRACT

Modifications in the sequence of Aequorea wild-type GFP provide products having markedly different excitation and emission spectra from corresponding products from wild-type GFP. In one class of modifications, the product derived from the modified GFP exhibits an alteration in the ratio of two main excitation peaks observed with the product derived from wild-type GFP. In another class, the product derived from the modified GFP fluoresces at a shorter wavelength than the corresponding product from wild-type GFP. In yet another class of modifications, the product derived from the modified GFP exhibits only a single excitation peak and enhanced emission relative to the product derived from wild-type GFP.

The invention was made with Government support under Grant No. NS27177,awarded by the National Institute of Health. The Government has certainrights in this invention.

This is a divisional application of U.S. application Ser. No.08/727,452, filed Oct. 18, 1996, which is a continuation ofPCT/US95/14692, filed Oct. 10, 1995, which is a continuation-in-part ofU.S. application Ser. No. 08/337,915, filed Nov. 10,1994, which hasissued as U.S. Pat. No. 5,625,048.

BACKGROUND OF THE INVENTION

This invention relates generally to the fields of biology and chemistry.More particularly, the invention is directed to modified fluorescentproteins and to methods for the preparation and use thereof.

In biochemistry, molecular biology and medical diagnostics, it is oftendesirable to add a fluorescent label to a protein so that the proteincan be easily tracked and quantified. The normal procedures for labelingrequires that the protein be covalently reacted in vitro withfluorescent dyes, then repurified to remove excess dye and any damagedprotein. If the labeled protein is to be used inside cells, it usuallyhas to be microinjected; this is a difficult and time-consumingoperation that cannot be performed on large numbers of cells. Theseproblems may, however, be eliminated by joining a nucleotide sequencecoding for the protein of interest with the sequence for a naturallyfluorescent protein, then expressing the fusion protein.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoriais a remarkable protein with strong visible absorbance and fluorescencefrom a p-hdroxybenzylideneimidazolone chromophore, which is generated bycyclization and oxidation of the protein's own Ser-Tyr-Gly sequence atpositions 65 to 67. A cDNA sequence SEQ ID NO:1! for one isotype of GFPhas been reported Prasher, D. C. et al,, Gene 111, 229-233 (1992)!;cloning of this cDNA has enabled GFP expression in different organisms.The finding that the expressed protein becomes fluorescent in cells froma wide variety of organisms Chalfie, M. et al., Science 263, 802-805(1994)! makes GFP a powerful new tool in molecular and cell biology andindicates that the oxidative cyclization must be either spontaneous ordependent only on ubiquitous enzymes and reactants.

A major question in protein photophysics is how a single chromophore cangive widely different spectra depending on its local proteinenvironment. This question has received the most attention with respectto the multiple colors of visual pigments based on retinal Merbs, S. L.& Nathans, J. Science 258, 46-466 (1992)!, but is also important in GFP.The GFP from Aequorea and that of the sea pansy Renilla reniformis sharethe same chromophore, yet Aequorea GFP has two absorbance peaks at 395and 475 nm, whereas Renilla GFP has only a single absorbance peak at 498nm, with about 5.5 fold greater monomer extinction coefficient than themajor 395 nm peak of the Aequorea protein Ward, W. W. in Bioluminescenceand Chemiluminescence (eds. DeLuca, M.A. & McElroy, W. D.) 235-242(Academic Press, New York, 1981)!. The spectra of the isolatedchromophore and denatured protein at neutral pH do not match the spectraof either native protein Cody, C. W. et al., Biochemistry 32, 1212-1218(1993)!.

For many practical applications, the spectrum of Renilla GFP would bepreferable to that of Aequorea, because wavelength discriminationbetween different fluorophores and detection of resonance energytransfer are easier if the component spectra are tall and narrow ratherthan low and broad. Furthermore, the longer wavelength excitation peak(475 nm) of Aequorea GFP is almost ideal for fluorescein filter sets andis resistant to photobleaching, but has lower amplitude than the shorterwavelength peak at 395 nm, which is more susceptible to photobleachingChalfie et al. (1994), supra!. For all these reasons, it would clearlybe advantageous to convert the Aequorea GFP excitation spectrum to asingle peak, and preferably at longer wavelengths.

There is also a need in the art for proteins which fluoresce atdifferent wavelengths. Variants of fluorescent proteins with differentcolors would also be very useful for simultaneous comparisons ofmultiple protein fates, developmental lineages, and gene expressionlevels.

Accordingly, it is an object of the present invention to provideimproved fluorescent proteins which do not suffer from the drawbacks ofnative Aequorea GFP.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has been determined thatparticular modifications in the polypeptide sequence of an Aequoreawild-type GFP SEQ ID NO:2! lead to formation of products having markedlydifferent excitation and emission spectra from corresponding productsderived from wild-type GFP. Visibly distinct colors and/or increasedintensities of emission make these products useful in a wide variety ofcontexts, such as tracking of differential gene expression and proteinlocalization.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood with reference to theaccompanying drawings, in which:

FIG. 1 compares different versions of GFP by gel electrophoresis andCoomassie blue staining;

FIG. 2 illustrates a proposed biosynthetic scheme for GFP;

FIGS. 3a and 3b illustrate the excitation and emission spectra ofwild-type and a first group of mutant GFPs;

FIGS. 4a and 4b illustrate the excitation and emission spectra ofwild-type and a second group of mutant GFPs;

FIG. 5 illustrates the rate of fluorophore formation in the wild-typeGFP and the Ser 65→Thr mutant;

FIGS. 6a and 6b illustrate the behavior of wild-type GFP and the Ser65→Thr mutant, respectively, upon progressive irradiation withultraviolet light; and

FIG. 7 illustrates fluorescence excitation and emission spectra of athird group of GFP mutants.

DETAILED DESCRIPTION OF THE INVENTION

GFP was expressed in E. coli under the control of a T7 promoter forquantitative analysis of the properties of the recombinant protein. Gelelectrophoresis under denaturing conditions showed protein of theexpected molecular weight (27 kDa) as a dominant band (FIG. 1), whichcould be quantified simply by densitometry of staining with Coomassieblue. Soluble recombinant GFP proved to have identical spectra and thesame or even slightly more fluorescence per mole of protein as GFPpurified from Aequorea victoria, showing that the soluble protein in E.coli undergoes correct folding and oxidative cyclization with as high anefficiency as in the jellyfish.

The bacteria also contained inclusion bodies consisting of proteinindistinguishable from jellyfish or soluble recombinant protein ondenaturing gels (FIG. 1). However, this material was completelynon-fluorescent, lacked the visible absorbance bands of the chromophore,and could not be made fluorescent even when solubilized and subjected toprotocols that renature GFP Ward, W. W. & Bokman, S. H., Biochemistry21, 4535-4540 (1982); Surpin, M. A. & Ward, W. W., Photochem. Photobiol.49, Abstract, 25S (1989)!. Therefore, protein from inclusion bodiesseemed permanently unable to generate the internal chromophore. Aninteresting intermediate stage in protein maturation could be generatedby growing the bacteria anaerobically. The soluble protein again lookedthe same as GFP on denaturing gels (FIG. 1) but was non-fluorescent. Inthis case, fluorescence gradually developed after admission of air, evenwhen fresh protein synthesis was blocked using puromycin andtetracycline. Evidently, the soluble non-fluorescent protein synthesizedunder anaerobic conditions was ready to become fluorescent onceatmospheric oxygen was readmitted. The fluorescence per protein moleculeapproached its final asymptotic value with a single-exponential timecourse and a rate constant of 0.24±0.06 hr⁻¹ (at 22° C.) measured eitherin intact cells with protein-synthesis inhibitors or in a lysate inwhich the soluble proteins and cofactors were a thousand fold moredilute. Such pseudo-first order kinetics strongly suggest that noenzymes or cofactors are necessary for the final step of fluorophoreformation in GFP.

It has thus been determined that formation of the final fluorophorerequires molecular oxygen and proceeds in wild-type protein with a timeconstant of ˜4 h at 22° C. and atmospheric pO₂. This was independent ofdilution, implying that the oxidation does not require enzymes orcofactors.

A molecular interpretation is presented in FIG. 2. If the newlytranslated apoprotein (top left) evades precipitation into inclusionbodies, the amino group of Gly 67 might cyclize onto the carbonyl groupof Ser 65 to form an imidazolidin-5-one, where the process would stop(top center) if O₂ is absent. The new N=C double bond would be expectedto promote dehydrogenation to form a conjugated chromophore;imidazolidin-5-ones are indeed known to undergo autoxidative formationof double bonds at the 4-position Kjaer, A. Acta Chem. Scand. 7,1030-1035 (1953); Kidwai, A. R. & Devasia, G. M. J. Org. Chem. 27,4527-4531 (1962)!, which is exactly what is necessary to complete thefluorophore (upper right). The protonated and deprotonated species(upper and lower right) may be responsible for the 395 and 470-475 nmexcitation peaks, respectively. The excited states of phenols are muchmore acidic than their ground states, so that emission would come onlyfrom a deprotonated species.

The Aequorea GFP cDNA was subjected to random mutagenesis byhydroxylamine treatment or polymerase chain reaction. Approximately sixthousand bacterial colonies on agar plates were illuminated withalternating 395 and 475 nm excitation and visually screened for alteredexcitation properties or emission colors.

According to a first aspect of the present invention, modifications areprovided which result in a shift in the ratio of the two excitationspeaks of the product after oxidation and cyclization relative to thewild type. Three mutants were found with significant alterations in theratio of the two main excitation peaks (Table I). The mutations weresequenced and recombined with the wild-type gene in different ways toeliminate neutral mutations and assign the fluorescence effects tosingle amino acid substitutions, except for H9 where two neighboringmutations have not yet been separated. They all lay in the C terminalpart of the protein (Table I), remote in primary sequence from thechromophore formed from residues 65-67.

These and other modifications are defined herein with reference to theamino acid sequence SEQ ID NO:2! encoded by the reported cDNA SEQ IDNO:1!; the first amino acid identified is the one found at the indicatedlocation in the reported sequence, while the second indicates thesubstitution found in the modified form. The fluorescent product derivedfrom a wild-type or modified GFP polypeptide sequence is no longerstrictly speaking a simple polypeptide after oxidation and cyclization;however, reference is sometimes made for sake of simplicity herein tothe polypeptide (e.g., "wild-type GFP" or "modified GFP") where what isintended would be obvious from the context. Compared with wild-type GFP,H9 (Ser 202→Phe, Thr 203→Ile) had increased fluorescence at 395 nmexcitation; P9 (Ile 167→Val) and P11 (Ile 167→Thr) were more fluorescentat 475 nm excitation.

One possibility for these spectral perturbations in P9 and P11 is thatthe mutations at Ile 167 shift a positive charge slightly closer to thephenolic group of the fluorophore; this should both increase thepercentage of phenolic anion, which is probably the species responsiblefor the 470-475 nm excitation peak, and shift the emission peakhypsochromically. However, the hypothesized ionizable phenolic groupwould have to be buried inside the protein at normal pH, because theratio of 471 to 396 nm peaks in the mutants could not be furtheraffected by external pH until it was raised to 10, just below thethreshold for denaturation. The pH-sensitivity of wild-type GFP issimilar Ward, W. W. et al., Photochem. Photobiol. 35, 803-808 (1982)!.

According to another aspect of the invention, a mutant P4 (Tyr 66→His)was identified which was excitable by ultraviolet and fluoresced brightblue in contrast to the green of wild type protein. The excitation andemission maxima were hypsochromically shifted by 14 and 60 nmrespectively from those of wild-type GFP. The mutated DNA was sequencedand found to contain five amino acid substitutions, only one of whichproved to be critical: replacement of Tyr 66 in the center of thechromophore by His (corresponding to a change in the GFP cDNA sequenceSEQ ID NO:1! at 196-198 from TAT to CAT).

The surprising tolerance for substitution at this key residue promptedfurther site-directed mutagenesis to Trp and Phe at this position. Trpgave excitation and emission wavelengths intermediate between Tyr andHis (Table I) but was only weakly fluorescent, perhaps due toinefficiency of folding or chromophore formation due to stericconsiderations. Phe gave weak fluorescence with an excitation maximum at358 nm and an emission maximum at 442 nm. Accordingly, pursuant to thisaspect of the invention modified GFP proteins which fluoresce atdifferent wavelengths (preferably, different by at least 10 nm and morepreferably, by at least 50 nm) relative to the native protein areprovided, for example, those wherein Tyr 66 is replaced by Phe, His orTrp.

In a further embodiment of this aspect of the invention, a double mutantY66H, Y145F was identified which had almost the same wavelengths as thesingle mutant Y66H but almost twice the brightness, due mainly to ahigher quantum efficiency of fluorescence. The double mutant alsodeveloped its fluorescence during overnight growth, whereas the singlemutant required several days.

In accordance with further embodiments of this aspect of the invention,a first round of mutagenesis to increase the brightness of Y66W yieldedM153T/V163A/N212K as additional substitutions. This mutant was subjectedto another round of mutagenesis, resulting in two further sets, N146Iand I123V/Y145H/H148R (Table II). The quantum efficiency of thesemutants is now comparable to wild-type GFP. The clustering of thesubstitutions in residues 145 to 163 suggest that those residues lierelatively close to the chromophore and that reductions in the size oftheir side chains might be compensating for the larger size oftryptophan compared to tyrosine.

Pursuant to yet another aspect of the present invention, modified GFPproteins are provided which provide substantially more intensefluorescence per molecule than the wild type protein. Modifications atSer 65 to Ala, Ieu, Cys, Val, Ile or Thr provide proteins withred-shifted and brighter spectra relative to the native protein. Inparticular, the Thr mutant (corresponding to a change in the GFP cDNAsequence SEQ ID NO:1! at 193-195 from TCT to ACT) and Cys mutant(corresponding to a change in the GFP cDNA sequence SEQ ID NO:1! at193-195 from TCT to TGT) are about six times brighter than wild typewhen excited at the preferred long-wavelength band above 450 nm. As aconsequence, these modified proteins are superior to wild type proteinsfor practically all applications. Further, the brightness of thesemodified proteins matches the brightness reported in the literature forRenilla GFP; thus, these proteins clearly obviate the objections to thedimness of Aequorea GFP. In fact, it is speculated that the chromophoresin these modified proteins may exhibit the optimum brightness whichcould be achieved with a general structure derived from the Aequorea GFPchromophore. In particular, these mutations provide products exhibitingone or more of the following salient characteristics which distinguishthem clearly over the corresponding product from a wild-type GFP:reduced efficiency of excitation by wavelengths between about 350 and420 nm; enhanced excitation and emission efficiency when excited withwavelengths longer than about 450 nm; increased resistance tolight-induced shifts in the excitation spectrum; and faster kinetics offluorophore generation. In contrast, mutations to Trp, Arg, Asn, Phe andAsp did not provide improved brightness.

Mutagenesis of S65T to shift its wavelengths further to the red yieldedM153A/K238E (Table II) as the GFP variant with the longest-wavelengthexcitation maximum yet described, 504 nm vs. 490 nm for S65T.Surprisingly, the emission peak hardly changed (514 nm vs. 511 nm), sothat the separation between the excitation and emission peaks (Stokes'shift) is extremely narrow, only 10 nm. This is one of the smallestvalues reported for any fluorophore in aqueous solution at roomtemperature. As in the Y66W series, M153 seems to be influential. It isdoubtful that K238E is important, because this substitution has beenfound to be without effect in other mutants.

As would be readily apparent to those working in the field, to providethe desired fluorescent protein it would not be necessary to include theentire sequence of GFP. In particular, minor deletions at either end ofthe protein sequence are expected to have little or no impact on thefluorescence spectrum of the protein. Therefore, by a mutant orwild-type GFP sequence for purposes of the present invention arecontemplated not only the complete polypeptide and oligonucleotidesequences discussed herein, but also functionally-equivalent portionsthereof (i.e., portions of the polypeptide sequences which exhibit thedesired fluorescence properties and oligonucleotide sequences encodingthese polypeptide sequences). For example, whereas the chromophoreitself (position 65-67) is obviously crucial, the locations of knownneutral mutations suggest that amino acids 76-115 are less critical tothe spectroscopic properties of the product. In addition, as would beimmediately apparent to those working in the field, the use of varioustypes of fusion sequences which lengthen the resultant protein and servesome functional purpose in the preparation or purification of theprotein would also be routine and are contemplated as within the scopeof the present invention. For example, it is common practice to addamino acid sequences including a polyhistidine tag to facilitatepurification of the product proteins. As such fusions do notsignificantly alter the salient properties of the molecules comprisingsame, modified GFPs as described herein including such fusion sequencesat either end thereof are also clearly contemplated as within the scopeof the present invention.

Similarly, in addition to the specific mutations disclosed herein, it iswell understood by those working in the field that in many instancesmodifications in particular locations in the polypeptide sequence mayhave no effect upon the properties of the resultant polypeptide. Unlikethe specific mutations described in detail herein, other mutationsprovide polypeptides which have properties essentially or substantiallyindistinguishable from those of the specific polypeptides disclosedherein. For example, the following substitutions have been found to beneutral (i.e., have no significant impact on the properties of theproduct): Lys 3→Arg; Asp 76→Gly; Phe 99→Ile; Asn 105→Ser; Glu 115→Val;Thr 225→Ser; and Lys 238→Glu. These equivalent polypeptides (andoligonucleotide sequences encoding these polypeptides) are also regardedas within the scope of the present invention. In general, thepolypeptides and oligonucleotide sequences of the present invention (inaddition to containing at least one of the specific mutations identifiedherein) will be at least about 85 % homologous, more preferably at leastabout 90% homologous, and most preferably at least about 95% homologous,to the wild-type GFP described herein. Because of the significantdifference in properties observed upon introduction of the specifiedmodifications into a GFP sequence, the presence of the specifiedmodifications relative to the corresponding reported sequence forwild-type GFP SEQ ID NO:2! are regarded as central to the invention.

The oligonucleotide sequences of the present invention are particularlyuseful in processes for labelling polypeptides of interest, e.g., by theconstruction of genes encoding fluorescent fusion proteins. Fluorescencelabeling via gene fusion is site-specific and eliminates the presentneed to purify and label proteins in vitro and microinject them intocells. Sequences encoding the modified GFPs of the present invention maybe used for a wide variety of purposes as are well known to thoseworking in the field. For example, the sequences may be employed asreporter genes for monitoring the expression of the sequence fusedthereto; unlike other reporter genes, the sequences require neithersubstrates nor cell disruption to evaluate whether expression has beachieved. Similarly, the sequences of the present invention may be usedas a means to trace lineage of a gene fused thereto during thedevelopment of a cell or organism. Further, the sequences of the presentinvention may be used as a genetic marker; cells or organisms labeled inthis manner can be selected by, e.g., fluorescence-activated cellsorting. The sequences of the present invention may also be used as afluorescent tag to monitor protein expression in vivo, or to encodedonors or acceptors for fluorescence resonance energy transfer. Otheruses for the sequences of the present invention would be readilyapparent to those working in the field, as would appropriate techniquesfor fusing a gene of interest to an oligonucleotide sequence of thepresent invention in the proper reading frame and in a suitableexpression vector so as to achieve expression of the combined sequence.

The availability of several forms of GFP with such different spectralproperties should facilitate two-color assessment of differential geneexpression, developmental fate, or protein trafficking. For example, ifone wanted to screen for a drug that is specific to activate expressionof gene A but not gene B, one could fuse the cDNA for one color of GFPto the promoter region of gene A and fuse the cDNA for another color tothe promoter region of gene B. Both constructs would be transfected intotarget cells and the candidate drugs could be assayed to determine ifthey stimulate fluorescence of the desired color, but not fluorescenceof the undesired color. Similarly, one could test for the simultaneousexpression of both A and B by searching for the presence of both colorssimultaneously.

As another example, to examine the precise temporal or spatialrelationship between the generation or location of recombinant proteinsX and Y within a cell or an organism, one could fuse genes for differentcolors of GFP to the genes for proteins X and Y, respectively. Ifdesired, DNA sequences encoding flexible oligopeptide spacers could beincluded to allow the linked domains to function autonomously in asingle construct. By examining the appearance of the two distinguishablecolors of fluorescence in the very same cells or organisms, one couldcompare and contrast the generation or location of the proteins X and Ywith much greater precision and less biological variability than if onehad to compare two separate sets of cells or organisms, each containingjust one color of GFP fused to either protein X or Y. Other examples ofthe usefulness of two colors would be obvious to those skilled in theart.

The further mutations to brighten the Y66H and Y66W variants of GFPenhance the possibility of using two or three colors of fluorescentprotein to track differential gene expression, protein localizations orcell fates. For example, mutants P4-3 (Y66H/Y145F), W7(Y66W/N146I/M153T/V163A/N212K) and S65T can all be distinguished fromeach other. P4-3 is specifically detected by exciting at 290-370 nm andcollecting emission at 420-460 nm. W7 is specifically detected byexciting at 410-457 mn and collecting emission at 465-495 nm. S65T isspecifically detected by exciting at 483-493 nm and collecting emissionat wavelengths greater than 510 nm. Bacteria carrying these threeproteins are readily discriminated under a microscope using the abovewavelength bandpass filters.

The chromophore in GFP is well buried inside the rest of the protein, somuch of the dimness of the original point mutants was presumably due tosteric mismatch between the substituted amino acid and the cavityoptimized for tyrosine. The location of the beneficial mutations impliesthat residues 145-163 are probably close to the chromophore. TheM153A/S65T mutant has the longest wavelengths and smallest Stokes'shiftof any known fluorescent protein that does not use a cofactor.

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe invention as defined by the claims appended hereto.

EXAMPLE 1

The coding region of GFP clone 10.1 Prasher et al. (1992), supra! wasamplified by PCR to create NdeI and BamHI sites at the 5'and 3'ends,respectively, and was cloned behind the T7 promoter of pGEMEX2 (Promega)replacing most of the T7 gene 10. The resulting plasmid was transformedinto the strain JM109(DE3) (Promega Corp., Madison, Wis.), and highlevel expression was achieved by growing the cultures at 24° C. tosaturation without induction by IPTG. To prepare soluble extracts, 1.5ml cell suspension were collected, washed and resuspended in 150 μl 50mM Tris/HCl, pH 8.0, 2 mM EDTA. Lysozyme and DNAse I were added to 0.2mg/ml and 20 μg/ml, respectively, and the samples were incubated on iceuntil lysis occurred (1-2 hours). The lysates were then clarified bycentrifuging at 12,000×g for 15 minutes. Inclusion bodies were obtainedas described in the literature Sambrook, J. et al. in Molecular Cloning:A Laboratory Manual Vol. 2, 17.37-17.41 (Cold Spring Harbor Press, ColdSpring Harbor, New York, 1989)!.

As illustrated in FIG. 1, soluble extracts of E. coli expressing GFPshow a predominant band which is absent in extracts from control cellsand has the same electrophoretic mobility as native GFP isolated fromthe jellyfish A. Victoria. Inclusion bodies of expressing cells consistmainly of non-fluorescent GFP which has the same mobility as solubleGFP. Non-fluorescent soluble GFP of anaerobically grown cultures is alsoa major band with correct mobility. Soluble extracts of the mutatedclones H9, P9, P11 and P4 again contain a dominant protein withessentially the same molecular weight.

Random mutagenesis of the GFP cDNA was done by increasing the error rateof the polymerase chain reaction with 0.1 mM MnCl₂, 50 μM dATP and 200μM of dGTP, dCTP, and dTTP Muhlrad, D. et al., Yeast 8, 79-82 (1992)!.The product was ligated into pGEMEX2 and subsequently transformed intoJM109(DE3). Colonies on agar were visually screened for differentemission colors and ratios of brightness when excited at 475 vs. 395 nm.

FIGS. 3a and 3b illustrate the excitation and emission spectra ofwild-type and mutant GFPs. In FIGS. 3a and 3b, -- wild-type; - - S202F,T203I; - - - I167T; - - - - - Y66W; -- Y66H. Samples were solublefractions from E. coli expressing the proteins at high level, except forY66W, which was obtained in very low yield and measured on intact cells.Autofluorescence was negligible for all spectra except those of Y66W,whose excitation spectrum below 380 nm may be contaminated byautofluorescence. Excitation and emission spectra were measured with 1.8nm bandwidths and the non-scanning wavelength set to the appropriatepeak. Excitation spectra were corrected with a rhodamine B quantumcounter, while emission spectra (except for Y66W) were corrected formonochromator and detector efficiencies using manufacturer-suppliedcorrection spectra. All amplitudes have been arbitrarily normalized to amaximum value of 1.0. A comparison of brightness at equal proteinconcentrations is provided in Table I.

                  TABLE I                                                         ______________________________________                                        Characteristics of mutated vs. wild-type GFP                                                   Excitation Emission Relative.sup.c                           Variant                                                                             Mutation   Maxima (nm).sup.a                                                                        Maxima (nm).sup.b                                                                      Fluorescence                             ______________________________________                                        Wild  none       396 (476)  508 (503)                                                                              (≡100%)                            type                                                                          H9    Ser 202→Phe,                                                                      398        511      117%.sup.d                                     Thr 203→Ile                                                      P9    Ile 167→Val                                                                       471 (396)  502 (507)                                                                              166%.sup.e                               P11   Ile 167→Thr                                                                       471 (396)  502 (507)                                                                              188%.sup.e                               P4    Tyr 66→His                                                                        382        448       57%.sup.f                               W     Tyr 66→Trp                                                                        458        480      n.d.                                     ______________________________________                                         .sup.a Values in parentheses are loweramplitude peaks.                        .sup.b Primary values were observed when exciting at the main excitation      peak; values in parentheses were observed when illuminating at the            loweramplitude excitation peak.                                               .sup.c Equal amounts of protein were used based on densitometry of gels       stained with Coomassie Blue (Fig. 1).                                         .sup.d Emission maxima of spectra recorded at excitation 395 nm were          compared.                                                                     .sup.e Emission maxima of spectra recorded at excitation 475 nm were          compared.                                                                     .sup.f Emission spectrum of P4 recorded at 378 nm excitation was              integrated and compared to the integrated emission spectrum of wild type      recorded at 475 nm excitation; both excitation and emission                   characteristics were corrected.                                          

EXAMPLE 2

Oligonucleotide-directed mutagenesis at the codon for Ser-65 of GFP cDNAwas performed by the literature method Kunkel, T. A. (1985) Proc. Natl.Acad. Sci. USA 82, 488! using the Muta-Gene Phagemid in VitroMutagenesis Kit version 2, commercially available from Bio-Rad,Richmond, Calif. The method employs a bacterial host strain deficientfor dUTPase (dut) and uracil-N-glycosylase (ung), which results in anoccasional substitution of uracil for thymine in newly-synthesized DNA.When the uracil-containing DNA is used as a wild-type template foroligonucleotide-directed in vitro mutagenesis, the complementary(mutant) strand can be synthesized in the presence of deoxynucleotides,ligase and polymerase using the mutagenic oligonucleotide to prime DNAsynthesis; the Version 2 kit utilizes unmodified T7 DNA polymerase tosynthesize the complementary strand. When the heteroduplex molecule istransformed into a host with an active uracil-N-glycosylase (whichcleaves the bond between the uracil base and the ribose molecule,yielding an apyrimidic site), the uracil-containing wild-type strand isinactivated, resulting in an enrichment of the mutant strand.

The coding region of GFP cDNA was cloned into the BamHI site of thephagemid pRSET_(B) from Invitrogen (San Diego, Calif.). This constructwas introduced into the dut, ung double mutant E. coli strain CJ236provided with the Muta-Gene kit and superinfected with helper phageVCSM13 (Stratagene, La Jolla, Calif.) to produce phagemid particles withsingle-stranded DNA containing some uracils in place of thymine. Theuracil-containing DNA was purified to serve as templates for in vitrosynthesis of the second strands using the mutagenic nucleotides asprimers. The DNA hybrids were transformed into the strain XL1blue(available from Stratagene), which has a functionaluracil-N-glycosylase; this enzyme inactivates the parent wild-type DNAstrand and selects for mutant clones. DNA of several colonies wereisolated and checked for proper mutation by sequencing.

To express the mutant proteins, the DNA constructs obtained bymutagenesis were transformed into E. coli strain BL21(DE3)LysS (Novagen,Madison, Wis.), which has a chromosomal copy of T7 polymerase to driveexpression from the strong T7 promotor. At room temperature 3 mlcultures were grown to saturation (typically, overnight) withoutinduction. Cells from 1 ml of culture were collected, washed and finallyresuspended in 100 μl of 50 mM Tris pH 8.0, 300 mM NaCl. The cells werethen lysed by three cycles of freeze/thawing (liquid nitrogen/30° C.water bath). The soluble fraction was obtained by pelletting cell debrisand unbroken cells in a microfuge.

To facilitate purification of the recombinant proteins, the vector usedfuses a histidine tag (6 consecutive His) to the N-terminus of theexpressed proteins. The strong interaction between histidine hexamersand Ni²⁺ ions permitted purification of the proteins by NI-NTA resin(available commercially from Qiagen, Chatsworth, Calif.). Microcolumns(10 μl bed volume) were loaded with 100 μl soluble extract (in 50 mMTris pH 8.0, 300 mM NaCl), washed with 10 bed volumes of the same bufferand with 10 volumes of the buffer containing 20 mM imidazole. Therecombinant proteins were then eluted with the same buffer containing100 mM imidazole.

Aliquots of the purified mutant GFP proteins were run along withwild-type GFP on a denaturing polyacrylamide gel. The gel was stainedwith Coomassie blue and the protein bands were quantified by scanning ona densitometer. Based on these results, equal amounts of each version ofprotein were used to run fluorescence emission and excitation spectra.

FIGS. 4a and 4b compare the excitation and emission spectra of wild-typeand Ser 65 mutants. In FIG. 4a, --S65T; - - S65A; - - - S65C; --wild-type (emission at 508 nm). In FIG. 4B, -- S65T; - - S65A; - - -S65C;  wild-type (excitation at 395 mn); -- wild-type (excitationat 475 nm). Excitation and emission spectra were measured with 1.8 nmbandwidths and the non-scanning wavelength set to the appropriate peak.As is apparent from FIG. 4b, all three mutants exhibited substantiallyhigher intensity of emission relative to the wild-type protein.

FIG. 5 illustrates the rates of fluorophore formation in wild-type GFPand in the Ser 65→Thr mutant. E. coli expressing either wild-type ormutant GFP were grown anaerobically. At time=0, each sample was exposedto air; further growth and protein synthesis were prevented bytransferring the cells to nutrient-free medium also containing sodiumazide as a metabolic inhibitor. Fluorescence was subsequently monitoredas a function of time. For each culture, the fluorescence intensitiesare expressed as a fraction of the final fluorescence intensity obtainedat t=18 to 20 hours, after oxidation had proceeded to completion. FromFIG. 5, it is apparent that development of fluorescence proceeds muchmore quickly in the mutant than in wild-type GFP, even afternormalization of the absolute brightnesses (FIGS. 4a and 4b). Therefore,when the development of GFP fluorescence is used as an assay forpromotor activation and gene expression, the mutant clearly gives a morerapid and faithful measure than wild-type protein.

FIGS. 6a and 6b illustrate the behavior of wild-type GFP and the Ser65→Thr mutant, respectively, upon progressive irradiation withultraviolet light. Numbers indicate minutes of exposure to illuminationat 280 nm; intensity was the same for both samples. Wild-type GFP (FIG.6a) suffered photoisomerization, as shown by a major change in the shapeof the excitation spectrum. Illumination with broad band (240-400 nm) UVcaused qualitatively similar behavior but with less increase ofamplitude in the 430-500 nm region of the spectrum. Thephotoisomerization was not reversible upon standing in the dark. Thisphotoisomerization would clearly be undesirable for most uses ofwild-type GFP, because the protein rapidly loses brightness when excitedat its main peak near 395 nm. The mutant (FIG. 6b) showed no suchphotoisomerization or spectral shift.

EXAMPLE 3

GFP cDNAs encoding for Tyr66→His (Y66H), Tyr66→Trp (Y66W), or Ser65→Thr(S65T) were separately further mutagenized by the polymerase chainreaction and transformed into E. coli for visual screening of colonieswith unusual intensities or colors. Isolation, spectral characterization(Table II and FIG. 7), and DNA sequencing yielded several additionaluseful variants.

Random mutagenesis of the gfp cDNA was done by increasing the error rateof the PCR with 0.1 mM MnCl₂ and unbalanced nucleotide concentrations.The GFP mutants S65T, Y66H and Y66W had been cloned into the BamH1 siteof the expression vector pRSETB (Invitrogen), which includes a T7promoter and a polyhistidine tag. The GFP coding region (shown in bold)was flanked by the following 5' and 3' sequences: 5'-G GAT CCC CCC GCTGAA TTC ATG . . . AAA TAA TAA GGA TCC-3'. The 5' primer for themutagenic PCR was the T7 primer matching the vector sequence; the 3'primer was 5'-GGT AAG CTT TTA TTT GTA TAG TTC ATC CAT GCC-3', specificfor the 3' end of GFP, creating a HindIII restriction site next to thestop codon. Amplification was over 25 cycles (1 min at 94° C., 1 min 52°C., 1 min 72° C.) using the AmpliTaq polymerase from Perkin Elmer. Fourseparate reactions were run in which the concentration of a differentnucleotide was lowered from 200 μM to 50 μM. The PCR products werecombined, digested with BamHI and HindIII and ligated to the pRSETB cutwith BamHI and HindIII. The ligation mixture was dialyzed against water,dried and subsequently transformed into the bacterial strain BL21(DE3)by electroporation (50 μl electrocompetent cells in 0.1 cm cuvettes,1900 V, 200 ohm, 25 μF). Colonies on agar were visually screened forbrightness as previously described herein. The selected clones weresequenced with the Sequenase version 2.0 kit from Unites StatesBiochemical.

Cultures with freshly transformed cells were grown at 37° C. to anoptical density of 0.8 at 600 nm, then induced with 0.4 mMisopropylthiogalactoside overnight at room temperature. Cells werewashed in PBS pH 7.4, resuspended in 50 mM Tris pH 8.0, 300 mM NaCl andlysed in a French press. The polyhistidine-tagged GFP proteins werepurified from cleared lysates on nickel-chelate columns (Qiagen) using100 mM imidazole in the above buffer to elute the protein.

Excitation spectra were obtained by collecting emission at therespective peak wavelengths and were corrected by a Rhodamine B quantumcounter. Emission spectra were likewise measured at the respectiveexcitation peaks and were corrected using factors from the fluorometermanufacturer (Spex Industries, Edison, N.J.). In cleavage experimentsemission spectra were recorded at excitation 368 nm. For measuring molarextinction coefficients, 20 to 30 μg of protein were used in 1 ml of PBSpH 7.4. Quantum yields of wild-type GFP, S65T, and P4-1 mutants wereestimated by comparison with fluorescein in 0.1 N NaOH as a standard ofquantum yield 0.91 ed. Miller, J. N., Standards in FluorescenceSpectrometry (Chapman and Hall, New York, 1981)!. Mutants P4 and P4-3were likewise compared to 9-amino-acridine in water (quantum yield0.98). W2 and W7 were compared to both standards, which fortunately gaveconcordant results.

FIG. 7 illustrates the fluorescence excitation and emission spectra ofdifferent GFP mutants. All spectra were normalized to a maximal valueof 1. Each pair of excitation and emission spectrum is depicted by adistinct line style.

The fluorescence properties of the obtained GFP mutants are reported inTable II.

                  TABLE II                                                        ______________________________________                                        Fluorescence properties of GFP mutants                                                      Excitation                                                                             Emission                                                                             Extinct. Coeff.                                                                         Quantum                               Clone                                                                              Mutations                                                                              max (nm) max (nm)                                                                             (M.sup.-1 cm.sup.-1)                                                                    yield                                 ______________________________________                                        P4-3 Y66H     381      445    14,000    0.38                                       Y145F                                                                    W7   Y66W     433 (453)                                                                              475 (501)                                                                            18,000 (17,100)                                                                         0.67                                       N146I                                                                         M153T                                                                         V163A                                                                         N212K                                                                    W2   Y66W     432 (453)                                                                              480    10,000 (9,600)                                                                          0.72                                       I123V                                                                         Y145H                                                                         H148R                                                                         M153T                                                                         V163A                                                                         N212K                                                                    P4-1 S65T     504 (396)                                                                              514    14,500 (8,600)                                                                          0.54                                       M153A                                                                         K238E                                                                    ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 716 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..716                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTT48                            MetSerLysGlyGluGluLeuPheThrGlyValValProIleLeuVal                              151015                                                                        GAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAG96                            GluLeuAspGlyAspValAsnGlyHisLysPheSerValSerGlyGlu                              202530                                                                        GGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGC144                           GlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCys                              354045                                                                        ACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTC192                           ThrThrGlyLysLeuProValProTrpProThrLeuValThrThrPhe                              505560                                                                        TCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACAG240                           SerTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGln                              65707580                                                                      CATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGA288                           HisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArg                              859095                                                                        ACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTC336                           ThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluVal                              100105110                                                                     AAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATT384                           LysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIle                              115120125                                                                     GATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAAC432                           AspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsn                              130135140                                                                     TATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGA480                           TyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGly                              145150155160                                                                  ATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTT528                           IleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerVal                              165170175                                                                     CAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCT576                           GlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyPro                              180185190                                                                     GTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCG624                           ValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer                              195200205                                                                     AAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTA672                           LysAspProAsnGluLysArgAspHisMetValLeuLeuGluPheVal                              210215220                                                                     ACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATA716                               ThrAlaAlaGlyIleThrHisGlyMetAspGluLeuTyrLys                                    225230235                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 238 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerLysGlyGluGluLeuPheThrGlyValValProIleLeuVal                              151015                                                                        GluLeuAspGlyAspValAsnGlyHisLysPheSerValSerGlyGlu                              202530                                                                        GlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheIleCys                              354045                                                                        ThrThrGlyLysLeuProValProTrpProThrLeuValThrThrPhe                              505560                                                                        SerTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGln                              65707580                                                                      HisAspPhePheLysSerAlaMetProGluGlyTyrValGlnGluArg                              859095                                                                        ThrIlePhePheLysAspAspGlyAsnTyrLysThrArgAlaGluVal                              100105110                                                                     LysPheGluGlyAspThrLeuValAsnArgIleGluLeuLysGlyIle                              115120125                                                                     AspPheLysGluAspGlyAsnIleLeuGlyHisLysLeuGluTyrAsn                              130135140                                                                     TyrAsnSerHisAsnValTyrIleMetAlaAspLysGlnLysAsnGly                              145150155160                                                                  IleLysValAsnPheLysIleArgHisAsnIleGluAspGlySerVal                              165170175                                                                     GlnLeuAlaAspHisTyrGlnGlnAsnThrProIleGlyAspGlyPro                              180185190                                                                     ValLeuLeuProAspAsnHisTyrLeuSerThrGlnSerAlaLeuSer                              195200205                                                                     LysAspProAsnGluLysArgAspHisMetValLeuLeuGluPheVal                              210215220                                                                     ThrAlaAlaGlyIleThrHisGlyMetAspGluLeuTyrLys                                    225230235                                                                     __________________________________________________________________________

What is claimed is:
 1. A composition of matters comprising:a fluorescentmodified form of an Aequorea wild-type GPP polypeptide, characterized inthat upon oxidation and cyclization of amino acid residues in saidfluorescent modified form corresponding to positions 65 to 67 ofwild-type GFP polypeptide sequence (SEQ ID NO:2) said fluorescentmodified form exhibits a different excitation and/or emission spectrumfrom a corresponding product of said wild-type GFP polypeptide sequence,with the proviso that when said fluorescent modified form comprises amutation at S65, said mutation at S65 is selected from the groupconsisting of S65A, S65C, S65T, S65L, S65V, and S65I.
 2. The compositionaccording to claim 1,wherein said fluorescent modified form exhibits andalteration in the ration of two main excitation peaks relative to saidwild-type GFP polypeptide sequence.
 3. The composition according toclaim 2,wherein said fluorescent modified form exhibits increasedfluorescence at a shorter-wavelength peak of the two main excitationpeaks than said wild-type GFP polypeptide sequence.
 4. The compositionaccording to claim 3,wherein said fluorescent modified form comprises areplacement of Ser at a position corresponding to position 202 in saidwild-type GFP polypeptide sequence by Phe and a replacement of Thr at aposition corresponding to position 203 by Ile.
 5. The compositionaccording to claim 2,wherein said fluorescent modified form exhibitsincreased fluorescence at a longer-wavelength peak of the two mainexcitation peaks than said wild-type GFP polypeptide sequence.
 6. Thecomposition according to claim 5,wherein said fluorescent modified formcomprises a replacement of Ile at a position corresponding to position167 of said wild-type GFP polypeptide sequence by Val or Thr.
 7. Thecomposition according to claim 5,wherein said fluorescent modified formcomprises a replacement of Ser at a position corresponding to position65 of said wild-type GFP sequence by Thr, a replacement of Met atposition 153 with Ala, and a replacement of Lys at position 238 withGlu.
 8. The composition according to claim 1,wherein said fluorescentmodified form fluoresces at a shorter wavelength than said wild-type GFPpolypeptide sequence.
 9. The composition according to claim 8,whereinsaid fluorescent modified form comprises a replacement of Tyr at aposition corresponding to position 66 of said wild-type GFP polypeptidesequence by Phe, His or Trp.
 10. The composition according to claim8,wherein said fluorescent modified form comprises a replacement of Tyrat a position corresponding to position 66 of said wild-type GFPpolypeptide sequence by His and a replacement of Tyr at position 145with Phe.
 11. The composition according to claim 8,wherein saidfluorescent modified form comprises a replacement of Tyr at a positioncorresponding to position 66 of said wild-type GFP polypeptide sequenceby Trp, a replacement of Asn at position 146 by Ile, a replacement ofMet at position 153 by Thr, a replacement of Val at position 163 by Ala,and a replacement of Asn at position 212 by Lys.
 12. The compositionaccording to claim 8,wherein said fluorescent modified form comprises areplacement of Tyr at a position corresponding to position 66 of saidwild-type GFP polypeptide sequence by Trp, a replacement of Ile atposition 123 by Val, a replacement of Tyr at position 145 by His, areplacement of His at position 148 by Arg, a replacement of Met atposition 153 by Thr, a replacement of Val at position 163 by Ala, and areplacement of Asn at position 212 by Lys.
 13. The composition accordingto claim 1,wherein said fluorescent modified form exhibits enhancedemission relative to said wild-type GFP polypeptide sequence.
 14. Thecomposition according to claim 13,wherein said fluorescent modifiedcomprises a replacement of Ser at a position corresponding to position65 of said wild-type GFP polypeptide sequence by an amino acid selectedfrom the group consisting of Ala, Cys, Thr, Leu, Val and Ile.
 15. Thecomposition according to claim 14,wherein said amino acid is Cys of Thr.16. A functional mutant fluorescent protein, comprising:a protein withan amino acid sequence that differs from an amino acid sequence of anAequorea wild type green fluorescent protein (SEQ ID NO:2) by at leastone amino acid substitution that is at position 65, wherein said atleast one substitution is either S65A, S65C, S65T, S65L, S65V, or S651,wherein said functional mutant fluorescent protein has an excitation oremission different from an excitation spectrum or emission spectrum ofsaid Aequorea wild type green fluorescent protein.
 17. The functionalmutant fluorescent protein of claim 16,wherein said at least one aminoacid substitution that is at position 65 is S65A.
 18. The functionalmutant fluorescent protein of claim 17,wherein said functional mutantfluorescent protein comprises a fusion protein.
 19. The functionalmutant fluorescent protein of claim 16,wherein said at least one aminoacid substitution that is at position 65 is S65C.
 20. The functionalmutant fluorescent protein of claim 19,wherein said functional mutantfluorescent protein comprises a fusion protein.
 21. The functionalmutant fluorescent protein of claim 16,wherein said at least one aminoacid substitution that is at position 65 is S65T.
 22. The functionalmutant fluorescent protein of claim 21,wherein said functional mutantfluorescent protein comprises a fusion protein.
 23. The functionalmutant fluorescent protein of claim 16,wherein said at least one aminoacid substitution that is at position 65 is S65L.
 24. The functionalmutant fluorescent protein of claim 23,wherein said functional mutantfluorescent protein comprises a fusion protein.
 25. The functionalmutant fluorescent protein of claim 16,wherein said at least one aminoacid substitution that is at position 65 is S65V.
 26. The functionalmutant fluorescent protein of claim 25,wherein said functional mutantfluorescent protein comprises a fusion protein.
 27. The functionalmutant fluorescent protein of claim 16,wherein said at least one aminoacid substitution that is at position 65 is S65I.
 28. The functionalmutant fluorescent protein of claim 27,wherein said functional mutantfluorescent protein comprises a fusion protein.
 29. The functionalmutant fluorescent protein of claim 16,wherein said functional mutantfluorescent protein consists of mutations S65T, M153A, and K238E. 30.The functional mutant fluorescent protein of claim 29,wherein saidfunctional mutant fluorescent protein comprises a fusion protein. 31.The functional mutant fluorescent protein of claim 16,wherein saidfunctional mutant fluorescent protein further comprises an amino acidsequence which targets said protein to the specific cellular locations.32. A functional mutant fluorescent protein, comprising:a protein withan amino acid sequence that differs from an amino acid sequence of anAequorea wild type green fluorescent protein (SEQ ID NO:2) by at leastone amino acid substitution that is at position 66, wherein said atleast one substitution is either Y66H or Y66W, further wherein saidfunctional mutant fluorescent protein has an excitation or emissiondifferent from an excitation spectrum or emission spectrum of saidAequorea wild type green fluorescent protein.
 33. The functional mutantfluorescent protein of claim 32,wherein said at least one amino acidsubstitution that is at position 66 is Y66W.
 34. The functional mutantfluorescent protein of claim 33,wherein said functional mutantfluorescent protein comprises a fusion protein.
 35. The functionalmutant fluorescent protein of claim 32,wherein said at least one aminoacid substitution that is at position 66 is Y66H.
 36. The functionalmutant fluorescent protein of claim 35,wherein said functional mutantfluorescent protein comprises a fusion protein.
 37. The functionalmutant fluorescent protein of claim 32,wherein said functional mutantfluorescent protein consists of mutations Y66H and Y145F.
 38. Thefunctional mutant fluorescent protein of claim 37,wherein saidfunctional mutant fluorescent protein comprises a fusion protein. 39.The functional mutant fluorescent protein of claim 32,wherein saidfunctional mutant fluorescent protein consists of mutations Y66W, N146I,M153T, V163A and N212K.
 40. The functional mutant fluorescent protein ofclaim 39,wherein said functional mutant fluorescent protein comprises afusion protein.
 41. The functional mutant fluorescent protein of claim32,wherein said functional mutant fluorescent protein consists ofmutations Y66W, I123V, Y145H, H148R, M153T, V163A, and N212K.
 42. Thefunctional mutant fluorescent protein of claim 41,wherein saidfunctional mutant fluorescent protein comprises a fusion protein.
 43. Afunctional mutant fluorescent protein, comprising:a protein with anamino acid sequence that differs from an amino acid sequence of anAequorea wild type green fluorescent protein (SEQ ID NO:2) by at leastone amino acid substitution in the region consisting of positions 65 and66, wherein said functional mutant fluorescent protein has an excitationor emission different from an excitation spectrum or emission spectrumof said Aequorea wild type green fluorescent protein.
 44. The functionalmutant fluorescent protein of claim 43,wherein said functional mutantfluorescent protein exhibits an alteration in the ratio of two mainexcitation peaks relative to Aequorea wild type green fluorescentprotein.
 45. The functional mutant fluorescent protein of claim44,wherein said functional mutant fluorescent protein exhibits increasedfluorescence at the shorter-wavelength peak of said two main excitationpeaks.
 46. The functional mutant fluorescent protein of claim 43,furthercomprising an amino acid substitution that is at position
 202. 47. Thefunctional mutant fluorescent protein of claim 46,wherein said aminoacid substitution that is at position 202 is S202F.
 48. The functionalmutant fluorescent protein of claim 43,further comprising an amino acidsubstitution that is at position
 203. 49. The functional mutantfluorescent protein of claim 48,wherein said amino acid substitutionthat is at position 203 is T203I.
 50. The functional mutant fluorescentprotein of claim 43,further comprising an amino acid substitution thatis at position
 167. 51. The functional mutant fluorescent protein ofclaim 50,wherein said amino acid substitution that is at position 167 isI167 V or I167T.
 52. The functional mutant fluorescent protein of claim43,further comprising an amino acid substitution that is at position153.
 53. The functional mutant fluorescent protein of claim 52,whereinsaid amino acid substitution that is at position 153 is M153T or M153A.54. The functional mutant fluorescent protein of claim 43,furthercomprising an amino acid substitution that is at position
 238. 55. Thefunctional mutant fluorescent protein of claim 54,wherein said aminoacid substitution that is at position 238 is K238E.
 56. The functionalmutant fluorescent protein of claim 43,further comprising an amino acidsubstitution that is at position
 145. 57. The functional mutantfluorescent protein of claim 56,wherein said amino acid substitutionthat is at position 145 is Y145H or Y145F.
 58. The functional mutantfluorescent protein of claim 43,further comprising an amino acidsubstitution that is at position
 146. 59. The functional mutantfluorescent protein of claim 58,wherein said amino acid substitutionthat is at position 146 is N146I.
 60. The functional mutant fluorescentprotein of claim 43,further comprising an amino acid substitution thatis at position 163 or
 148. 61. The functional mutant fluorescent proteinof claim 60,wherein said amino acid substitution that is at position 163is V163A and said amino acid substitution at position 148 is H148R. 62.The functional fluorescent protein of claim 43,further comprising anamino acid substitution that is at position 212 or
 123. 63. Thefunctional fluorescent protein of claim 62,wherein said amino acidsubstitution that is at position 212 is N212K and said amino acidsubstitution at position 123 is I123V.
 64. The functional fluorescentprotein of claim 43,wherein said functional fluorescent proteincomprises a fusion protein.